Method for introducing siRNA into cells by photochemical internalisation

Abstract

The present invention relates to a method for introducing an siRNA molecule into the cytosol of a cell, said method comprising i) contacting said cell with an siRNA molecule, a carrier and a photosensitizing agent, and ii) irradiating the cell with light of a wavelength effective to activate the photosensitizing agent, wherein said carrier comprises a cationic polyamine such as a lipopolyamine in a non-liposomal formulation, polyethyleneimine (PEI), a betacyclodextrin amine polymer, an amine group containing dendrimer, and a cationic peptide. Cells or a population of cells obtainable by the method, a composition containing an siRNA molecule and the carrier molecule, kits and therapeutic uses of the above are also provided.

The invention claimed is: 
     
       1. A method for introducing an siRNA molecule into the cytosol of a cell, said method comprising
 i) contacting said cell with an siRNA molecule, a carrier and a photosensitizing agent; and 
 ii) irradiating the cell with light of a wavelength effective to activate the photosensitizing agent, 
 wherein said carrier comprises a cationic polyamine selected from
 (a) a branched polyethyleneimine (PEI) with a molecular weight of from 1.2 to 1.8 kDa, wherein the N/P ratio of the siRNA and PEI used in said method is from 5 to 300, and wherein said PEI has an M n  (number average molecular weight) value of 500-1500 by gel permeation chromatography (GPC), and 
 (b) a cationic peptide selected from poly-Arginine and a copolymer of L or D arginine, wherein said cationic peptide is at least 70,000 Da in molecular weight. 
 
 
     
     
       2. The method of  claim 1  wherein the siRNA molecule is 12-28 nucleotides long. 
     
     
       3. The method of  claim 1  wherein said cell is a mammalian cell. 
     
     
       4. The method of  claim 1 , further comprising the additional step of contacting said siRNA with said carrier. 
     
     
       5. The method of  claim 1  wherein the siRNA molecule and the carrier molecule are contacted with one another for 20-40 minutes before being contacted with the cell. 
     
     
       6. The method of  claim 1 , wherein 10 nM-200 nM siRNA is used for transfection. 
     
     
       7. The method of  claim 1  wherein a photosensitizer carrier selected from a polycation, polyethyleneimine, a dendrimer, a cationic lipid and a peptide is additionally present. 
     
     
       8. The method of  claim 1 , wherein the siRNA is mixed with the carrier so as to form a complex, which is then administered to the cell simultaneously or sequentially with the photosensitizing agent. 
     
     
       9. The method of  claim 1  wherein said method is performed by contacting said cell with a photosensitizing agent, contacting said cell with the carrier and the siRNA molecule to be introduced and irradiating said cell with light of a wavelength effective to activate the photosensitizing agent, wherein said irradiation is performed prior to the cellular uptake of said siRNA molecule and said carrier into an intracellular compartment containing said photosensitizing agent. 
     
     
       10. A method of inhibiting the expression of a target gene comprising introducing an siRNA molecule into a cell containing said target gene by the method of  claim 1 , wherein said siRNA molecule specifically inhibits expression of said target gene. 
     
     
       11. A method of treating or preventing a disease, disorder or infection in a patient comprising introducing an siRNA molecule and carrier into one or more cells in vitro, in vivo or ex vivo according to the method of  claim 1  and where necessary administering said cells to said patient. 
     
     
       12. The method of  claim 11  wherein the disease to be treated is typified by abnormal gene expression or which would benefit from suppression of one or more genes. 
     
     
       13. The method of  claim 11  wherein the siRNA is mixed with the carrier so as to form a complex, which complex is then introduced to the one or more cells simultaneously with the photosensitizing agent. 
     
     
       14. The method of  claim 11  wherein the siRNA is mixed with the carrier so as to form a complex, which complex is then introduced to the one or more cells sequentially with the photosensitizing agent. 
     
     
       15. The method of  claim 12 , wherein the disease is cancer. 
     
     
       16. The method of  claim 1  wherein the photosensitizing agent is selected from a porphyrin, phthalocyanine, purpurin, chlorin, benzoporphyrin, lysomotropic weak base, naphthalocyanine, cationic dye, tetracycline, 5-aminolevulinic acid or an ester thereof, or a derivative or a pharmaceutically acceptable salt of any said photosensitizing agent. 
     
     
       17. The method of  claim 16  wherein the photosensitizing agent is sulfonated tetraphenylporphine (TPPS) TPPS 4 , TPPS 2a , tetrasulfonated aluminium phthalocyanine (ALPcS) AlPcS 2a , sulfonated meso-tetraphenyl chlorin (TPCS) TPCS 2a , 5-aminolevulinic acid or an ester or pharmaceutically acceptable salt thereof. 
     
     
       18. A combination of (a) a photosensitizing agent and (b) a composition that comprises an siRNA molecule and a carrier molecule wherein the carrier molecule comprises a cationic polyamine selected from (i) a branched polyethyleneimine (PEI) with a molecular weight of from 1.2 to 1.8 kDa, wherein the N/P ratio of the siRNA and PEI in said composition is from 5 to 300, and wherein said PEI has an M n  (number average molecular weight) value of 500-1500 by gel permeation chromatography (GPC), and (ii) a cationic peptide selected from poly-Arginine and a copolymer of L or D arginine, wherein said cationic peptide is at least 70,000 Da in molecular weight. 
     
     
       19. The combination of  claim 18  wherein the composition and the photosensitizing agent are formulated for simultaneous, separate, or sequential use in therapy.